The inherent limitations of normal fluorescence have encouraged the development of alternative techniques and compounds. There are at least two such limitations on using normal fluorescent molecules for biological imaging and therapy. First, cells have many fluorescent molecules of their own, leading to unwanted background fluorescence that can be particularly problematic in sensitive experiments such as single-molecule imaging. Second, the intense light typically used by microscopes to excite fluorescent molecules can damage cells, especially light at the ultraviolet (UV) and blue end of the visible spectrum. Photo damage may also occur indirectly, if the excited state of a fluorescent molecule reacts with a nearby molecule instead of emitting fluorescence.
Photodynamic therapy (PDT) is a favorable cancer treatment modality due to its minimally invasive nature, leading to fewer side effects than chemotherapy and less damage to marginal tissue. Photodynamic therapy is a treatment that uses a non-toxic photosensitive compound, called a photosensitizer or photosensitizing agent, and a particular type of light. When photosensitizers are exposed to a specific wavelength of light, they produce a form of oxygen that kills nearby cells. In air and tissue, molecular oxygen occurs in a triplet state, whereas most other molecules are in a singlet state. Reactions between these are forbidden by quantum mechanics, thus oxygen is relatively non-reactive at physiological conditions. A photosensitizer is a chemical compound that can be promoted to an excited state upon absorption light and undergo intersystem crossing with oxygen to produce singlet oxygen. This species rapidly reacts with organic compounds it encounters, and is thus highly cytotoxic.
Each photosensitizer is activated by light of a specific wavelength. The photosensitizers used in conventional photodynamic therapy are mostly activated by visible light, which cannot pass through thick tissue. The application of these visible light photosensitizers is limited to treating tumors on or just under the skin or on the lining of internal organs or cavities and is less effective when treating large tumors deep under the skin. Treatment methods involve directly activating a photosensitizer to generate singlet oxygen that is toxic to cancer cells under irradiation with light in the visible region. Of the colors in this spectrum, longer wavelength red (i.e., 620-670 nm) light is preferred by the majority of photosensitizers in clinical practice due to its improved tissue penetration compared to shorter wavelength light. The use of near-infrared light in photodynamic therapy can afford greater penetration depths than that of visible light because the absorbance for most biomolecules reaches a minimum in the near-infrared light window (having a wavelength of 700-1100 nm).
Upconverting nanoparticles (UCNPs) are a promising new generation of agents for bio imaging and photodynamic therapy. Normal fluorescence converts higher-energy (i.e., shorter wavelength) light to lower-energy (i.e., longer wavelength) emitted light. Upconversion luminescence, on the other hand, refers to an anti-Stokes type process in which the sequential absorption of two or more low-energy (i.e., longer wavelength) photons by a nanoparticle, is followed by the emission of a single higher-energy (i.e., shorter wavelength) photon. Applications of UCNPs include bio sensing, chemical sensing, in vivo imaging, drug delivery, photodynamic therapy and photoactivation.
Lanthanide-doped UCNPs are dilute guest—host systems where trivalent lanthanide ions are dispersed in an appropriate dielectric host lattice that typically has a dimension of less than about 100 nm. Lanthanide-doped UCNPs have been developed that are excited by tissue-penetrable near-infrared light and have emissions ranging from visible to ultraviolet light. Lanthanide ion (Ln3+) containing UCNPs are able to absorb near-infrared (NIR) photons and convert such low energy excitation into shorter wavelength emissions. Haase, et al., Angew. Chem. Mt. Ed., 2011 50, 5808. Due to the long lived energy levels of lanthanide ions, the intensity of the anti-Stokes luminescence of such UCNPs is more potent compared with that of conventional synthetic dyes. UCNPs have been developed that are excited by tissue-penetrable near-infrared light (e.g., 980 nm) and have emissions ranging from visible to ultraviolet.
Lanthanide-doped upconverting nanoparticles are particularly useful for use in photodynamic therapy, an emerging treatment modality for a variety of diseases. In contrast to other photosensitizers such as photofrin, low cost 5-aminolevulinic acid (5-ALA) has unique advantages due to its hydrophilicity, higher selectivity in cancerous cells, and reduced concomitant photosensitivity, leading to minimal trauma in surrounding tissue. 5-ALA converts to the photosensitizer protoporphyrin IX (PpIX) via a heme biosynthesis pathway. Because this occurs to a greater extent in tumors than in non-cancerous cells due to the down regulation of the enzyme ferrochelatase (a PpIX degrading factor) in cancerous cells, PpIX selectively targets cancerous cells over non-cancerous cells. Peng, et al., 5-Aminolevulinic Acid-Based Photodynamic Therapy, Cancer, 1997, 79, 2282. Under irradiation with red light, PpIX converts triplet oxygen into singlet oxygen, inducing cell death. Despite much progress in its clinical use, ALA's application is limited because while red light offers the maximum tissue penetration of the wavelengths in PpIX's activation spectrum, it is still absorbed or dispersed by common components of tissue, rendering deep-tissue (>1 cm) photodynamic therapy challenging.